Na/K-ATPase (NKA) is the main Na extrusion pathway and therefore is essential in [Na]i regulation. In the heart, [Na]i critically modulates [Ca]i and contractility via Na/Ca exchange (NCX), which makes understanding [Na]i regulation extremely important. Phospholemman (PLM or FXYD1) is a transmembrane protein of the FXYD family of proteins that are known to associate with and modulate NKA. PLM is the only FXYD protein abundant in the heart, where it is a major substrate for phosphorylation by protein kinase A (PKA) and C (PKC). During the initial award period, we showed that PLM regulates NKA similar to the way phospholamban (PLB) regulates SR Ca-ATPase (SERCA). That is, PLM inhibits NKA by reducing its [Na]i affinity, and PLM phosphorylation relieves this inhibition. We also showed that PLM phosphorylation and the consequent NKA stimulation is integral to the sympathetic fight or flight response (tempering the rise in [Na]i and cellular Ca load, and limiting Ca overload-induced arrhythmias). We found that PLM physically associates with NKA 1PLM phosphorylation (via immunoprecipitation), but in fluorescence resonance energy transfer (FRET) measurements, that PLM phosphorylation alters the PLM-NKA interaction and that PLM forms homo- oligomers. All of this resembles SERCA-PLB. Despite its physiological significance, the mechanism of PLM- NKA interaction is poorly understood. Thus, the overall goal of this renewal proposal is to decipher mechanistically how PLM and NKA interact functionally. Here we will combine FRET, [Na]i and [Ca]i measurements, patch-clamp and molecular biology techniques in cardiac myocytes and HEK 293 cells. Aim 1 focuses on the structure-function of the NKA-PLM (and PLM-PLM complex). This is important for several reasons. First, the recent NKA crystal structures with associated FXYDs suggest some sites on NKA and FXYDs that are within interaction distance (at least in the particular stable conformation in the crystals), but this requires testing in live cell membranes. That will be tested in Aim 1 by in situ FRET measurements, Co-IP and NKA activity measurements in cells. Second, it is unknown how PLM-PLM homo-oligomers interact (e.g. whether they form stable multimers with a structural basis like PLB) and whether there is a pool of PLM multimers in equilibrium with PLM-NKA dimers that exerts functional regulation (as for PLB-SERCA). That will be assessed in Aim 1 as well. Aim 2 focuses more narrowly on the intriguing possibility that PLB (at least PLB human mutants that are relevant to human disease) can be misdirected to the sarcolemma and regulate NKA. We will test whether WT PLB can also interact with and modulate NKA activity if it is in the sarcolemma (and conversely if PLM can interact functionally with SERCA if it is expressed in the ER/SR). Aim 3 examines questions that are inspired from our novel and surprising observation that ouabain abolishes FRET between NKA and either PLM or NKA. This will provide important information about how dimerization (hetero or homo) of NKA relates to its functional activity, and possibly also the role of NKA in kinase signaling cascades.